Project Summary Aedes aegypti is the principal mosquito vector of viruses that cause Zika, yellow fever, chikungunya, and dengue, the most widespread and significant arboviral disease in the world. Immature mosquitoes of this species are often concentrated in natural and artificial water-filled containers in urban environments and are susceptible to control efforts. Larviciding, the application of microbial or chemical agents to kill mosquito larvae before they are reproducing adults that transmit human disease pathogens, is a key component of integrated A. aegypti control strategies worldwide. However, given the frequent development of pesticide resistance and rising concerns about the off-target effects of pesticides, the current larvicide repertoire will soon reach its expiration date. It is therefore critical to identify new environmentally safe larvicidal agents for use in integrated A. aegypti control programs. Recent RNA interference (RNAi) studies have demonstrated that small interfering RNA (siRNA), which can be designed to be species-specific, can be used for selective targeting of A. aegypti larval genes. A recent high-throughput screen in our laboratory identified >100 larval lethal genes in A. aegypti. Although larval microinjection facilitated the screen, this technique cannot be used for RNAi in the field. The objective of the proposed investigation is to identify an effective and economical delivery system for species-specific interfering RNA larvicides targeting A. aegypti. This research program will test the hypothesis, which is supported by preliminary experiments, that interfering RNA produced and delivered in Saccharomyces cerevisiae (baker's yeast) can be utilized as mosquito larvicidal agents. S. cerevisiae, a model organism that is genetically tractable and inexpensive to culture, can be engineered to produce interfering RNA in the form of short hairpin RNA (shRNA), which is easily amplified through yeast cultivation. Yeast is both a strong odorant attractant and a source of nutrition for laboratory-bred A. aegypti larvae. Furthermore, yeast can be dried and packaged for storage and shipment, making it ideal for delivery to less economically developed countries. The aims of this investigation are to (1) express short hairpin RNA (shRNA) corresponding to A. aegypti larval lethal genes in S. cerevisiae and to (2) test this yeast interfering RNA delivery system in simulated- and semi-field experiments. This research program will generate necessary preliminary data for future proposals centered on conducting larger-scale semi-field and field studies at international field sites. These studies are requisite steps for advancement toward the long-term goal of including cost-effective species-specific yeast interfering RNA larvicides in integrated mosquito control programs.